In vitro and In vivo Inhibition of Plant Polyamine Oxidase Activity by Polyamine Analogues

MAIALE, SANTIAGO; MARINA, MARÍA; SÁNCHEZ, DIEGO H., PIECKENSTAIN, FERNANDO L. & RUIZ, OSCAR A.

PHYTOCHEMISTRY

PERGAMON-ELSEVIER SCIENCE LTD

Año: 2008 vol. 69 p. 2552 - 2556

0031-9422

Polyamine oxidase (PAO) from oat seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme showed a high affinity for spermidine and spermine (Km 5.5 mM and 1.2 mM respectively), and also oxidized norspermidine (Km 64 mM). Natural and synthetic diamines, cyclohexylamine (CHA), putrescine analogue 1-aminooxy-3-aminopropane (APA), and several polyamine analogues have proved to have an inhibitory effect on PAO activity and none of them was substrate. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. In contrast, APA showed mixed inhibition kinetics and a Ki value of 0.113 mM.  In addition, in vitro enzymatic activity assay showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene] (SL-11144); the tetramine 1,14-bis-ethylamino-5,10-diazatetradecane or bis-ethyl-homospermine, BHSPM (SL-11156) and the pentamine 1,19-bis-ethylamino-5,10,15-triazanonadecane, BE-4-4-4-4 (SL-11061) displayed a potent competitive inhibitory activity against PAO with Ki values 5.84 nM, 0.11 mM and 7.6 nM respectively, whereas CHA proved to be a weak competitive inhibitor with a Ki value of 0.5 mM.  These analogues did not inhibit the mycelial growth of fungus Sclerotinia sclerotiorum and bacteria Pseudomonas viridiflava in vitro, contrasting the inhibitory effect of putrescine analogue APA on both microorganisms previously observed in our lab.  Moreover, with concentration levels similar to the ones used for polyamine analogues, the guazatine (a well-known fungicide and at the same time, a PAO inhibitor) inhibited by 85% the S. sclerotiorum mycelial growth on Czapek-Dox medium. Finally, analogue SL-11061 inhibited the PAO activity observed in segments of maize leaves in vivo. The results obtained provide new insights into the research on the influence of PAO activity on plant biotic and abiotic stresses.